Pyrophosphorolysis-activated polymerization (PAP) using ribonucleic acid (RNA) template

ABSTRACT

A new method of RNA-PAP was developed that can directly amplify RNA template without additional treatment. RNA-PAP brings in a new mechanism for amplification of RNA template in which RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization are serially coupled using 3′ blocked primers. Due to this serial coupling, RNA-PAP has high selectivity against mismatches on the RNA template, providing highly specific amplification of RNA template. In addition, mutant polymerases were genetically engineered for higher efficiency of RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patentapplication Ser. No. 61/724,292, filed on Nov. 8, 2012.

SEQUENCE LISTING

This application is being filed along with a Sequence Listing and itselectronic format entitled SequenceListing.txt.

BACKGROUND

The present invention relates to the field of molecular biology andparticularly to methods for amplification of ribonucleic acid (RNA).

PAP Technology for Amplification of DNA Template

Pyrophosphorolysis activated polymerization is a method for nucleic acidamplification¹ ² where pyrophosphorolysis and polymerization areserially coupled by DNA polymerase using 3′ blocked primers. A primer isblocked at the 3′ end with a non-extendable nucleotide (3′ blocker),such as a dideoxynucleotide, and cannot be directly extended by DNApolymerase. When the 3′ blocked primer anneals to its complementary DNAtemplate, DNA polymerase can remove the 3′ blocker from the 3′ blockedprimer in the presence of pyrophosphate, which reaction is calledpyrophosphorolysis. The DNA polymerase can then extend the 3′ unblockedprimer on the DNA template. In addition to references cited herein, PAPhas been described in U.S. Pat. Nos. 6,534,269, 7,033,763, 7,105,298,7,238,480, 7,504,221, 7,914,995, and 7,919,253.

The serial coupling of pyrophosphorolysis and extension using the 3′blocked primer in PAP results in an extremely high selectivity¹ ³because a significant nonspecific amplification (Type II error) requiresmismatch pyrophosphorolysis followed by mis-incorporation by the DNApolymerase, an event with a frequency estimated to be 3.3×10⁻¹¹.

The bi-directional form of PAP (Bi-PAP) is especially suitable forallele-specific amplification that uses two opposing 3′ blocked primerswith a single-nucleotide overlap at their 3′ ends³ ⁴. Bi-PAP can detectone copy of a mutant allele in the presence of 10⁹ copies of thewildtype DNA without false positive amplifications.

PAP was initially tested with Tfl and Taq polymerases using DNA templateof the human dopamine D1 gene¹, proving the principle that DNA-dependentDNA pyrophosphorolysis and DNA-dependent DNA polymerization can beserially coupled. The efficiency of PAP was greatly improved usingTaqFS, a genetically engineered polymerase containing contain a F667Ymutation⁵, which were demonstrated using other DNA templates² ⁴ ⁶.

However, no evidence has showed that PAP can work using RNA template, along-felt but unsolved need. For PAP to work using RNA template, it isalso required RNA-dependent DNA polymerization and RNA-dependent DNApyrophosphorolysis which latter feasibility has not been demonstrated.

RNA-Dependent DNA Polymerization or Reverse Transcription Used in RT-PCR

The first step of RT-PCR is DNA polymerization or primer extension onRNA template that is catalyzed by RNA-dependent DNA polymerase orreverse transcriptase. Avian myeloblastosis virus (AMV) and moloneymurine leukemia virus (MMLV) reverse transcriptases, two mesophilicretroviral transcriptases, are commonly used in this first step toconvert the RNA template into its complimentary DNA (cDNA) product⁷ ⁸.Native Taq, a thermophilic DNA polymerase, has measurable reversetranscriptase activity particularly in the presence of Mn²⁺ divalention⁹. rTth, another thermophilic DNA polymerase, shows over 100-foldgreater reverse transcriptase activity than Taq even though they havesignificant amino acid sequence similarity¹⁰. Furthermore, Taq and rTthpolymerases were also genetically engineered in order for higher reversetranscriptase activity¹¹ ¹² ¹³ ¹⁴.

RNA-Dependent DNA Pyrophosphorolysis

Taq, Tfl, TaqFS, Pfu, and Vent polymerases can catalyze DNA-dependentDNA pyrophosphorolysis¹ ² ³ ¹⁵. HIV and HCV reverse transcriptases werealso reported to catalyze DNA-dependent DNA pyrophosphorolysis thatremoves 3′ dideoxynucleotide from DNA primer on synthetic DNA (ratherthan RNA) template¹⁶ ¹⁷.

However, there was no report of RNA-dependent DNA pyrophosphorolysis ofpolymerase that removes 3′ deoxyribonucleotide or 3′ dideoxynucleotideor 3′ acyclonucleotide from a primer using RNA as template.

Advantages of the Invention

It is advantageous that RNA-PAP can direct amplify RNA template withoutadditional treatment. In addition, RNA-PAP has high selectivity againstmismatches on the RNA template, providing highly specific amplificationof RNA template. Furthermore, we genetically engineered mutantpolymerases for higher RNA-PAP efficiency.

SUMMARY OF THE INVENTION

A new method of RNA-PAP was developed that can directly amplify RNAtemplate. RNA-PAP brings in a new mechanism for amplification of RNAtemplate in which RNA-dependent DNA pyrophosphorolysis and RNA-dependentDNA polymerization are serially coupled using 3′ blocked primers.

The RNA-PAP method for synthesizing nucleic acid using RNA templatecomprises:

(a) a 3′ blocked primer that has a non-extendable nucleotide at the 3′end (3′ blocker) anneals to its complementary RNA template, (b)RNA-dependent DNA pyrophosphorolysis removes the 3′ blocker from the 3′blocked primer to produce a 3′ unblocked primer, and (c) RNA-dependentDNA polymerization extends the 3′ unblocked primer.

Of the RNA-PAP method, the steps (a), (b), and (c) can be repeated untila desired level of the extended nucleic acid is achieved.

In an embodiment, the RNA-PAP method can further include a second primerin the reaction: (d) the second primer anneals to the nucleic acidproduct of the step (c), and (e) DNA-dependent DNA polymerizationextends the second primer, thereby an exponential amplification can beachieved.

In another embodiment, the RNA-PAP method can further include a second3′ blocked primer: (f) the second 3′ blocked primer anneals to thenucleic acid product of the step (c), (g) DNA-dependent DNApyrophosphorolysis removes the 3′ blocker from the blocked primer toproduce a 3′ unblocked primer, and (h) DNA-dependent DNA polymerizationextends the 3′ unblocked primer.

Of the RNA-PAP method, steps (b) and (c) are catalyzed by a polymerase.

In a preferred embodiment, steps (b), (c), (d), (e), (g), and (h) arecatalyzed by a polymerase in one reaction tube.

The polymerase may be a genetically engineered DNA polymerase selectedfrom the group consisting of TaqFS, TaqFS681G, TaqFS681K, TaqFS681V,TaqFS681Y, TaqFS608V681 G, TaqFS599V602A605A608V681G, TaqFS742S747Icorresponding to Taq polymerase according to SEQ ID NO: 10.

In a preferred embodiment, the genetically engineered polymerase isselected from the group of mutants consisting of 599V, 602A, 605A, 608V,667Y, 681G, 681K, 681V, 681R, 742S, and 747I mutations corresponding toTaq polymerase according to SEQ ID NO: 10.

In another preferred embodiment, the genetically engineered polymeraseis selected from group of mutants consisting of amino acids located at599, 602, 605, 608, 667, 681, 742, and 747 corresponding to Taqpolymerase according to SEQ ID NO: 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the principle of RNA-PAP. The DNA primer is blockedat the 3′ end with, e.g., a dideoxynucleotide, preventing it fromextended by polymerase. When the 3′ blocked primer anneals to itscomplementary RNA template, the 3′ dideoxynucleotide can be removed byRNA-dependent DNA pyrophosphorolysis. Then the unblocked DNA primer canbe extended on the RNA template by RNA-dependent DNA polymerization.

FIG. 2 illustrates optimal dNTP concentration for RNA-dependent DNApolymerization. RT-PCR Assays II (panels A to D) and III (panels E to H)amplified from 0.25 ng of total RNA in 25 μl of reaction. One Unit ofeach polymerases of Taq (panels A and E), rTth (panels B and F), TaqFS(panels C and G), and TaqFS742S747I (panels D and H) was used. Theamplification plot is showed with cycle number and SybrGreenfluorescence unit for the given cycle.

FIG. 3 illustrates RNA-PAP feasibility with various polymerases. RNA-PAPAssays IV (panels A to D), V (panels E to H) and VI (panels I to L)amplified from 0.25 ng of total RNA. For each assay, four polymerases ofTaqFS (panels A, E, and I), TaqFS681G (panels B, F, and J), TaqFS681K(panels C, G, and K), and TaqFS742S747I (panels D, H, and L) weretested. The amount of each enzyme was 2-fold serially diluted from 1 Uto 1/16 U in 25 μl of reaction.

DETAILED DESCRIPTION OF THE INVENTION Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art.

PCR refers to polymerase chain reaction.

RT-PCR refers to reverse transcription polymerase chain reaction.

Pyrophosphorolysis is the reverse reaction of deoxyribonucleic acidpolymerization. In the presence of pyrophosphate, the 3′ nucleotide isremoved by a polymerase from duplex DNA to generate a triphosphatenucleotide and a 3′ unblocked duplex DNA:[dNMP]_(n)+PPi→[dNMP]_(n-1)+dNTP¹⁸.

DNA-dependent DNA pyrophosphorolysis refers to pyrophosphorolysis of DNAusing DNA template. It is the reverse reaction of DNA-dependent DNApolymerization

RNA-dependent DNA pyrophosphorolysis refers to pyrophosphorolysis of DNAusing RNA template. It is the reverse reaction of RNA-dependent DNApolymerization.

Polymerase refers to a polymerase characterized as polymerization orextension of deoxyribonucleic acids.

DNA-dependent DNA polymerase refers to a polymerase characterized as DNApolymerization using DNA template.

RNA-dependent DNA polymerase or reverse transcriptase refers to apolymerase characterized as DNA polymerization using RNA template.

3′ blocked primer refers to an oligonucleotide with a 3′ non-extendablenucleotide (3′ blocker), such as a dideoxynucleotide. The 3′ nucleotidecould not be directly extended, but it can be removed bypyrophosphorolysis and then the unblocked primer can be extended bypolymerase.

PAP refers to pyrophosphorolysis activated polymerization.

RNA-PAP refers to PAP using RNA template.

cDNA refers to a complementary DNA molecule synthesized using RNAtemplate.

Thermostable refers to an enzyme that is heat stable or heat resistant.

Protein mutation refers to a change in amino acid residue at a locationof a protein, like Taq polymerase. The change in amino acid residue isdefined with respect to a naturally occurring protein. A protein havinga mutation is referred to as a “mutant” protein.

Unless indicated otherwise, an amino acid position should be interpretedas the analogous position in all DNA polymerases, even though the singleletter amino acid code specifically relates to the amino acid residue atthe indicated position in Taq polymerase.

Principle of RNA-PAP

The invention is a RNA amplification method of synthesizing desirednucleic acid on RNA template (FIG. 1).

The method comprises the following steps: a) hybridization: annealing tothe RNA template a complementary 3′ blocked primer that has a 3′non-extendable nucleotide (3′ blocker), b) RNA-dependent DNApyrophosphorolysis: removing the 3′ blocker from the 3′ blocked primer,and c) RNA-dependent DNA polymerization: extending the 3′ unblockedprimer to synthesize the desired nucleic acid (FIG. 1).

The method yields a single-stranded complementary DNA (cDNA), or anRNA/cDNA duplex. Subsequent to step c, the cDNA product can be furtheramplified, for example, by PAP or PCR.

The method may employ a polymerase that catalyzes both RNA-dependent DNApyrophosphorolysis and RNA-dependent DNA polymerization.

3′ blockers include but not limited to 3′ dideoxynucleotides. Other 3′blockers, such as acyclonucleotides that substitute a2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar³, mayalso be used.

A biological sample may contain RNA molecules. The RNA may beheterogeneous in which the target RNA is a small portion.

EXAMPLES

The practice of the present invention employs, unless otherwiseindicated, common techniques of chemistry, molecular biology, andrecombinant DNA, e.g., those by Sambrook et al., Molecular Cloning, 2ndEd.¹⁹; Sambrook and Russell, Molecular Cloning, 3rd Ed.²⁰; Ausubel etal., Current Protocols in Molecular Biology ²¹.

Example 1 Materials and Methods Preparation of Primers

Each 3′ blocked primer, 30 nucleotides long and blocked with ddCMP atthe 3′ end, was chemically synthesized and HPLC purified by IntegratedDNA Technologies. The regular DNA primers were also synthesized by thesame vendor (Table 1).

TABLE 1 List of primers and assays Product Sequence (5′ to 3′) sizeStarting Assay Name ^(b) (SEQ ID NO:) Location (bp) template I.P53(13280)20D AGGGTCCCCAGGCCTCTG Intron 208 Genomic PCR AT (1) 5 DNAP53(13488)22U GCCACTGACAACCACCCT Intron TAAC (2) 6 II. ACTB(426)25DCAACCGCGAGAAGATGA Exons  63 Total RT- CCCAGATC (3) 3 and 4 RNA PCR ^(a)ACTB(488)25U GCAACGTACATGGCTGG Exon 4 GGTGTTGA (4) III. ACTB(426)25DCAACCGCGAGAAGATGA Exons 280 Total RT- CCCAGATC (3) 3 and 4 RNA PCRACTB(705)25U TTCCCGCTCGGCCGTGGT Exon 4 GGTGAAG (5) IV. ACTB(426)25DCAACCGCGAGAAGATGA Exons  63 Total RNA- CCCAGATC (3) 3 and 4 RNA PAP ^(a)ACTB(488)30U GCAACGTACATGGCTGG Exon 4 GGTGTTGAAGGTddC (6) ^(c) V.ACTB(426)25D CAACCGCGAGAAGATGA Exons 147 Total RNA- CCCAGATC (3) 3 and 4RNA PAP ^(a) ACTB(572)30U ACAGTGTGGGTGACCCC Exon 4 GTCACCGGAGTCddC (7)VI. GAPDH(50)- ACCATGGGGAAGGTGAA Exon 2  61 Total RNA- 30DGGTCGGAGTCAAddC (8) RNA PAP ^(a) GAPDH(110)- TGGTGACCAGGCGCCCA Exon 330U ATACGACCAAATddC (9) ^(a) In order to avoid non-specificamplification from potentially contaminated genomic DNA, RT-PCR andRNA-PAP primers were designed to anneal to sequences of the transcriptthat span intron regions. ^(b) For example, P53 means the human P53gene; (13280), 5′ end of the primer begins at nucleotide 13280 accordingto GenBank accession: x54156; D, downstream (i.e., in the direction oftranscription). The ACTB mRNA is from GenBank accession: NM_001101.3.The GAPDH mRNA is from GenBank accession: AB062273.1. The precise sizesand locations of the PCR fragment can be obtained from the informativenames. ^(c) Due to the availability of chemical synthesis, ddC wasexampled.

Total RNA Extraction

The total RNA was extracted from blood white cells using QIAamp RNA kitaccording to Qiagen' protocol (QIAamp RNA Blood Mini Handbook). Withinthe process, RNase-Free DNase was used to remove contaminated genomicDNA. The concentration of the total RNA was measured by aspectrophotometer at 260 nm. The quality was controlled with A260/A280ratio between 1.8 and 2.1. It was stored at −20° C. until used.

Construction of Recombinant Plasmids

The amino acid sequence of the wildtype form of Taq polymerase is asdescribed in GenBank accession AAA27507.1 (SEQ ID NO: 10). The numberingstarts at the amino terminus residue. The letter is the single letteramino acid code for the amino acid residue at the indicated position.

Recombinant plasmid that encodes the wildtype form of Taq polymerase wasconstructed by ligating the Taq polymerase coding sequence, a 2.4 kb DNAsegment from 121 nt to 2619 nt of GenBank accession J04639, into pET14bvector at the Nde I and BamH I restriction sites according to NavagenpET system user manual. Then the recombinant plasmid transformed DH5α E.coli cells and its plasmid DNA was extracted. The ligated Taq polymerasecoding sequence was confirmed by ABI Sanger sequencing analysis.

Eight recombinant plasmids that encode mutant forms of Taq polymerasewere constructed using mutagenic primers designed by QuikChange primerdesign program and QuikChange lightning site-directed mutagenesis kitaccording to Stratagene's user manual. Then each mutated plasmidtransformed DH5α E. coli cells and its plasmid DNA was extracted. Themutant DNA sequence was confirmed by ABI Sanger sequencing analysis.

TaqFS contained G46D and F667Y mutations, TaqFS681G contained G46D,F667Y and E681G mutations, TaqFS681K contained G46D, F667Y and E681Kmutations, TaqFS681V contained G46D, F667Y and E681V mutations,TaqFS681Y contained G46D, F667Y and E681Y mutations, TaqFS608V681Gcontained G46D, 608V, F667Y and E681G mutations,TaqFS599V602A605A608V681G contained G46D, I599V, E602A, L605A, A608V,F667Y and E681G mutations, and TaqFS742S747I contained G46D, F667Y,E742S, and M747I mutations. Individual substitution mutations areindicated by the form of a letter/number/letter combination. The lettersare the single letter code for amino acid residues. The numbers indicatethe amino acid residue position of the mutation site according to SEQ IDNO: 10.

Expression and Extraction of Mutant Polymerases

The mutant Taq polymerases were expressed in transformed T7 ExpresslysY/I^(q) E. coli cells according to BioLabs' manual. Before the startcodon of Taq polymerase, 6× His-Tag residues were located andco-expressed. Because the expressed polymerase contained His-Tag at theN-terminus, it was purified using Qiagen Ni-NTA His-Tag technology. Atypical yield of the purified polymerase was 4 mg from 500 ml of inducedE. coli culture. SDS-PAGE analysis of the purified protein showed onemajor band of about 95,000 Daltons after Coomassie Blue staining,indicating ≧90% purity.

The enzyme was stored in the storage buffer containing 20 mM Tris-HCl(pH 8.0 at 25° C.), 100 mM KCl, 0.1 mM EDTA, 50% Glycerol, 0.5%Tween-20, and 0.5% NP-40 at −20° C. until use.

PCR to Calibrate DNA-Dependent DNA Polymerase Activity

The primers of Assay I were designed to amplify a 209-bp region of exon6 of the human P53 gene. Each PCR mixture contained a total volume of 25μl: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 200 μM each dNTPs(dATP, dTTP, dGTP and dCTP), 0.1 μM each primer, 0.1× SybrGreen I dye,0.02% Twee-20, 0.02% NP-40, 20 ng of genomic DNA, and various units ofpolymerase.

A Bio-Rad CFX96 real time PCR detection system was used forquantification of the amplified product. Analysis mode: SybrGreenfluorophore, Baseline setting: baseline subtracted curve fit, Thresholdcycle (Ct) determination: single threshold, Baseline method: SYBR autocalculated, Threshold setting: auto calculated.

The cycling entailed denaturation at 94° C. for 15 seconds, annealing at55° C. for 30 seconds, and elongation at 72° C. for 1 minute for 30cycles. Before thermocycling, a step of 94° C. for 2 minutes was appliedto completely denature the genomic DNA.

For the activity calibration, Taq polymerase (Roche) was taken asstandard and its amount was 2-fold serially diluted from 1 U to 1/16 Uin 25 μl of reaction. For comparison, other enzymes were diluted in thesame way. The minimum unit of a polymerase with a measurable Ct within30 cycles reflected its compatible level of DNA-dependent DNA polymeraseactivity.

To confirm the amplified product, melting curving analysis was followedfrom 68° C. to 95° C. with increment 0.5° C. and holding 5 seconds.

RT-PCR to Measure RNA-Dependent DNA Polymerization

Unless stated otherwise, the RT-PCR reaction mixture of 25 μl contained880 mM Tris-HCl (pH 8.0 at 25° C.), 10 mM (NH₄)₂SO₄, 1.2 mM MgCl₂, 25 μMeach dNTPs (dATP, dTTP, dGTP and dCTP), 0.1 μM each primer of Assay IIor III, 90 μM Na₄PP_(i), 0.1× SybrGreen I dye, 0.02% Twee-20, 0.02%NP-40, 1 U of polymerase, and 0.25 ng of total RNA template.

A Bio-Rad CFX96 real time PCR detection system was used forquantification of the amplified product. Analysis mode: SybrGreenfluorophore, Baseline setting: baseline subtracted curve fit, Thresholdcycle (Ct) determination: single threshold, Baseline method: SYBR autocalculated, Threshold setting: auto calculated.

The cycling entailed 96° C. for 12 seconds, 60° C. for 30 seconds, 64°C. for 30 seconds, and 68° C. for 30 seconds for a total of 30-35cycles. A denaturing step of 96° C. for 2 minutes was added before thefirst cycle.

To confirm the amplified product, melting curving analysis was followedfrom 68° C. to 95° C. with increment 0.5° C. and holding 5 seconds.

For further confirmation, the product was electrophoresed through astandard 3% agarose gel. The gel was stained with ethidium bromide forUV photography by a charge-coupled device camera.

RNA-PAP

Unless stated otherwise, the PAP reaction mixture of 25 μl contained 880mM Tris-HCl (pH 8.0 at 25° C.), 10 mM (NH₄)₂SO₄, 1.2 mM MgCl₂, 25 μMeach dNTPs (dATP, dTTP, dGTP and dCTP), 0.1 μM each primer of Assay IV,V, or VI, 90 μM Na₄PP_(i), 0.1× SybrGreen I dye, 0.02% Twee-20, 0.02%NP-40, various units of polymerase, and 0.25 ng of total RNA template.

A Bio-Rad CFX96 real time PCR detection system was used forquantification of the amplified product. Analysis mode: SybrGreenfluorophore, Baseline setting: baseline subtracted curve fit, Thresholdcycle (Ct) determination: single threshold, Baseline method: SYBR autocalculated, Threshold setting: auto calculated.

The cycling entailed 96° C. for 12 seconds, 60° C. for 30 seconds, 64°C. for 30 seconds, and 68° C. for 30 seconds for a total of 30-35cycles. A denaturing step of 96° C. for 2 min was added before the firstcycle.

To confirm the amplified product, melting curving analysis was followedfrom 68° C. to 95° C. with increment 0.5° C. and holding 5 seconds toconfirm the amplified product.

For further confirmation, the product was electrophoresed through astandard 3% agarose gel. The gel was stained with ethidium bromide forUV photography by a charge-coupled device camera.

Example 2 Optimal Conditions for RNA-Dependent DNA Polymerization

RT-PCR includes two steps of 1) reverse transcription of RNA templateinto cDNA product and 2) regular PCR amplification of the cDNA product.Reverse transcription is considered as the efficiency limiting reactionof the whole process.

In reverse transcription, AMV⁷ and MMLV⁸ reverse transcriptases as wellas Taq⁹, Tth¹⁰ polymerases and their genetically engineered forms¹¹ ¹²¹³ ¹⁴ are commonly used with each dNTP concentration to be 200 μm. InRT-PCR, this concentration of dNTPs is suitable to the reversetranscription step and the subsequent PCR step. However, it severelyinhibited pyrophosphorolysis¹. Thus, an optimal dNTP concentration wasneeded to explore.

To search for optimal reaction conditions, we designed two RT-PCR AssaysII and III (Table 1). RT-PCR Assay II amplified a 63-bp ACTB mRNA regionwith primers ACTB(426)25D and ACTB(488)25U, and Assay III amplified a280-bp ACTB mRNA region with primers ACTB(426)25D and ACTB(705)25U. Eachassay had a forward primer and a reverse primer with the reverse primermatching the RNA template.

First, we examined the effect of various dNTP concentrations in thepresence of Mg²⁺ divalent ion. In FIG. 2, three dNTP conditions of 25 μMeach dNTP with 90 μM PPi, 25 μM each dNTP without PPi, as well as 200 μMdNTP without PPi were tested. Of four polymerases of Taq, rTth, TaqFS,and Taq681G, one unit was added to 25 μl of reaction. The polymeraseactivity for DNA-dependent DNA polymerization was calibrated accordingto Taq's activity (Roche) by amplifying a 209-bp region of exon 6 of theP53 gene (PCR Assay I) from human genomic DNA under standard PCRconditions. In addition, one unit of Taq DNA polymerase is defined asthe amount of enzyme that will incorporate 10 nmol of dNTP intoacid-insoluble material in 30 minutes at 75° C. (Roche). With the twoRT-PCR Assays II and III, we found that the RNA-dependent DNA polymeraseactivity was higher with 25 μM each dNTP than with 200 μM. Thus, 25 μMrather than 200 μM each dNTP was used for the following testing.

Second, we examined the effect of various polymerase amounts. Eightpolymerases of TaqFS, TaqFS681G, TaqFS681K, TaqFS681V, TaqFS681Y,TaqFS608V681G, TaqFS599V602A605A608V681G were tested. The amount of eachenzyme was 2-fold serially diluted from 1 U to 1/16 U in 25 μl ofreaction (Table 2). The minimum unit of a polymerase with a measurableCt within 30 cycles was considered to reflect the compatible level ofRNA-dependent DNA polymerization because it is the efficiency limitingreaction of the whole process. We found that the eight enzymes showeddifferent levels of RNA-dependent DNA polymerase activity.

In addition, in melting curving analysis, only one melting peak showedthe T_(m) value from the amplified product (T_(m)=80.5-82° C. for AssaysII and T_(m)=89.5-90° C. for Assay III). No-template-negative controldid not show any measurable Ct.

Example 3 RNA-PAP

The process of RNA-PAP can be divided into two steps. In the first stepwith RNA as starting template, RNA-dependent DNA pyrophosphorolysisremoves the 3′ blocker from the 3′ blocked primer, and thenRNA-dependent DNA polymerization extends the 3′ unblocked primer togenerate a DNA product. In the second step with the DNA product astemplate, DNA-dependent DNA pyrophosphorolysis removes the 3′ blockerfrom the 3′ blocked primer and then DNA-dependent DNA polymerizationextends the 3′ unblocked primer. Thus, four distinct reactions areinvolved that can be catalyzed by a polymerase.

For proof of principle, we designed three RNA-PAP Assays IV, V, and VI(Table 1) that amplified from RNA template. Each assay had a forwardprimer and a reverse primer for exponential amplification. The reverseprimer, blocked by a dideoxynucleotide at the 3′ end, matched the RNAtemplate.

For each assay, eight polymerases of TaqFS, TaqFS681G, TaqFS681K,TaqFS681V, TaqFS681Y, TaqFS608V681G, TaqFS599V602A605A608V681G, andTaqFS742S747I were examined. The amount of each enzyme was 2-foldserially diluted from 1 U to 1/16 U in 25 μl of reaction.

RNA-PAP Assay VI amplified a 63-bp ACTB mRNA region with primersACTB(426)25D and ACTB(488)30U (Table 2, FIG. 3A). In the first step,RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide fromthe 3′ blocked primer ACTB(488)30U and then RNA-dependent DNApolymerization extended the unlocked primers by 34 bases.

RNA-PAP Assay V amplified a 147-bp ACTB mRNA region with primersACTB(426)25D and ACTB(572)30U (Table 2, FIG. 3B). In the first step,RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide fromthe 3′ blocked primer ACTB(572)30U and then RNA-dependent DNApolymerization extended the unlocked primers by 118 bases.

RNA-PAP Assay VI amplified a 61-bp GAPDH mRNA region with primersGAPDH(50)-30D and GAPDH(110)-30U (Table 2, FIG. 3C). In the first step,RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide fromthe 3′ blocked primer GAPDH(110)-30U and then RNA-dependent DNApolymerization extended the unlocked primers by 32 bases.

With Assays IV, V, and VI, we proved the principle of RNA-PAP andparticularly demonstrated the feasibility of 1) RNA-dependent DNApyrophosphorolysis and 2) the serial coupling of RNA-dependent DNApyrophosphorolysis and RNA-dependent DNA polymerization. We also foundthat five enzymes of TaqFS681K, TaqFS681V, TaqFS681Y, TaqFS608V681G, andTaqFS742S747I showed higher activities particularly in RNA-dependent DNApyrophosphorolysis.

In addition, in melting curving analysis, only one melting peak showedthe T_(m) value from the amplified product (T_(m)=80.5-81.5° C. forAssay IV, T_(m)=87.5-88° C. for Assay V, and T_(m)=82-83° C. for AssayVI). No-template-negative control did not show any measurable Ct.

TABLE 2 The minimum units of polymerases required for RT-PCR andRNA-PAP^(a) RT- RT- PCR PCR Assay RNA-PAP RNA-PAP RNA-PAP PolymeraseAssay II III Assay IV Assay V Assay VI TaqFS 0.125U^(b) 1U 0.5U 1U 0.5UTaqFS681G 0.25U NA 1U NA NA TaqFS681K 0.125U 0.25U 0.0625U 0.125U 0.125UTaqFS681V 0.125U 0.5U 0.25U 0.5U 0.25U TaqFS681Y 0.125U 0.5U 0.125U0.25U 0.25U TaqFS608V681G 0.125U 0.5U 0.125U 0.125U 0.0625UTaqFS599V602A NA^(c) NA 1U NA NA 605A608V 681G TaqFS742S747I 0.25U 0.5U0.25U 0.5U 0.25U ^(a)The unit is defined as DNA-dependent DNA polymeraseactivity calibrated according to Taq's activity. ^(b)with 0.25 ng oftotal RNA, the amount of enzyme was 2-fold serially diluted from 1U to1/16U in 25 μl of reaction. The minimum required unit of a polymerasewith a measurable Ct was counted within 30 cycles. ^(c)no Ct was calledwithin 30 cycles.RNA-Dependent DNA Pyrophosphorolysis vs. RNA-Dependent DNAPolymerization

The process of RNA-PAP can be divided into two steps. In the first stepwith RNA as starting template are two distinct reactions ofRNA-dependent DNA pyrophosphorolysis and RNA-dependent DNApolymerization catalyzed serially by a polymerase.

To show their relative importance, the minimum units of polymerases werecompared for RT-PCR and RNA-PAP (Table 3). In RT-PCR Assay II,RNA-dependent DNA polymerization extended 38 bases from the 3′ end ofthe primer ACTB(488)25U. On the other hand in RNA-PAP Assay IV,RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide fromthe 3′ blocked primer ACTB(488)30U and then RNA-dependent DNApolymerization extended the unlocked primers by 34 bases. In RNA-PAPAssay VI, RNA-dependent DNA pyrophosphorolysis removed the 3′ ddCnucleotide from the 3′ blocked primer GAPDH(110)-30U and thenRNA-dependent DNA polymerization extended the unlocked primers by 32bases.

TABLE 3 Ratio of minimum units required for RT-PCR and RNA-PAP Ratio ofRatio of Assay Assay RT-PCR RNA-PAP RNA-PAP II to II to Polymerase AssayII Assay IV Assay VI IV VI TaqFS 0.125U 0.5U 0.5U ¼ ¼ TaqFS681G 0.25U 1UNA ¼ TaqFS681K 0.125U 0.0625U 0.125U 2 1 TaqFS681V 0.125U 0.25U 0.25U ½½ TaqFS681Y 0.125U 0.125U 0.25U 1 ½ TaqFS608V681G 0.0625U 0.125U 0.0625U½ ½ TaqFS599V602A NA 1U NA 605A608V 681G TaqFS742S747I 0.25U 0.25U 0.25U1 1

Because of the small amplicon sizes, the ratios of minimum requiredunits between RT-PCR Assay II and RNA-PAP Assay IV or VI indicate therelative importance between RNA-dependent DNA pyrophosphorolysis andRNA-dependent DNA polymerization (Table 3). Polymerases with the ratiosof minimum units to be ≧1 are preferred, such as TaqFS681K, for RNA-PAP.

REFERENCES

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What is claimed is:
 1. A method for synthesizing nucleic acid on RNAtemplate, comprising: (a) annealing to the RNA template a complementary3′ blocked primer that has a non-extendable nucleotide at the 3′ end,(b) removing the 3′ non-extendable nucleotide from the 3′ blocked primerby pyrophosphorolysis to produce a 3′ unblocked primer, and (c)extending the 3′ unblocked primer to produce a nucleic acid product. 2.The method of claim 1, wherein the steps (a), (b), and (c) are repeatedone or more times.
 3. The method of claim 1, further comprising: (d)annealing a second primer to the nucleic acid product of the step (c),and (e) extending the second primer.
 4. The method of claim 1, furthercomprising: (f) annealing a second 3′ blocked primer having anon-extendable nucleotide at the 3′ end to the nucleic acid product ofthe step (c), (g) removing the 3′ non-extendable nucleotide from thesecond 3′ blocked primer by pyrophosphorolysis to produce a second 3′unblocked primer, and (h) extending the second 3′ unblocked primer. 5.The method of claim 1, wherein steps (b) and (c) are catalyzed by apolymerase.
 6. The method of claim 5, wherein the polymerase is selectedfrom the group consisting of TaqFS, TaqFS681G, TaqFS681K, TaqFS681V,TaqFS681Y, TaqFS608V681G, TaqFS599V602A605A608V681G, and TaqFS742S747Ipolymerases corresponding to Taq polymerase according to SEQ ID NO: 10.7. The method of claim 5, wherein the polymerase has at least onemutation selected from the group consisting of 599V, 602A, 605A, 608V,667Y, 681G, 681K, 681V, 681R, 742S, and 7471 mutations corresponding toTaq polymerase according to SEQ ID NO:
 10. 8. The method of claim 5,wherein the polymerase has at least one mutation at an amino acidresidue selected from the group of consisting of 599, 602, 605, 608,667, 681, 742, and 747 residues corresponding to Taq polymeraseaccording to SEQ ID NO:
 10. 9. The method of claim 1, wherein dATP,dTTP, dGTP, and dCTP, or their analogs are used as substrates for theextending.
 10. The method of claim 1, wherein pyrophosphate or itsanalogs are used as substrates for the pyrophosphorolysis.
 11. A methodfor analyzing RNA template, comprising: (a) annealing to the RNAtemplate a complementary primer, and (b) removing the 3′ nucleotide fromthe primer by pyrophosphorolysis.
 12. A method of claim 11, wherein theprimer has a non-extendable nucleotide at the 3′ end.
 13. A method ofclaim 11, further comprising: (c) extending the 3′ unblocked primer. 14.A polymerase for pyrophosphorolysis having at least one mutation,wherein the mutation is at an amino acid residue selected from the groupconsisting of 599, 602, 605, 608, 681, 742, and 747 residuescorresponding to Taq polymerase according to SEQ ID NO:
 10. 15. Thepolymerase of claim 14, wherein the mutation is at an amino acid residueselected from the group consisting of I599, E602, L605, A608, E681,E742, and M747 residues corresponding to Taq polymerase according to SEQID NO:
 10. 16. The polymerase of claim 14, wherein the mutation isselected from the group consisting of 599V, 602A, 605A, 608V, 681G,681K, 681V, 681R, 742S, and 7471 mutations corresponding to Taqpolymerase according to SEQ ID NO:
 10. 17. The polymerase of claim 14,wherein the mutation is selected from the group consisting of I599V,E602A, L605A, A608V, E681G, E681K, E681V, E681R, E742S, and M747Imutations corresponding to Taq polymerase according to SEQ ID NO: 10.